Release from persistent T cell receptor engagement and blockade of aryl hydrocarbon receptor activity enhance IL-6-dependent mouse follicular helper T-like cell differentiation in vitro

Follicular helper T (Tfh) cells are crucial for humoral immunity. Dysregulation of Tfh cell differentiation can cause infectious, allergic, and autoimmune diseases. To elucidate the molecular mechanisms underlying Tfh cell differentiation, we attempted to establish an in vitro mouse model of Tfh cell differentiation in the absence of other cell types. Various cytokines and cell surface molecules are suggested to contribute to the differentiation. We found that stimulating naïve CD4+ T cells with immobilized antibodies to CD3, ICOS, and LFA-1 in the presence of soluble anti-CD28 antibody, IL-6, and antibodies that block IL-2 signaling for 3 days induced the expression of Bcl6 and Rorc(γt), master regulator genes of Tfh and Th17 cells, respectively. TGF-β significantly enhanced cell proliferation and Bcl6 and Rorc(γt) expression. An additional 2 days of culture without immobilized antibodies selectively downregulated Rorc(γt) expression. These cells produced IL-21 and promoted B cells to produce IgG antibodies. Adding the aryl hydrocarbon receptor (AhR) antagonist CH-223191 to the T cell culture further downregulated Rorc(γt) expression without significantly affecting Bcl6 expression, and upregulated expression of a key Tfh marker, CXCR5. Although their CXCR5 expression levels were still not high, the CH-223191-treated cells showed chemotactic activity towards the CXCR5 ligand CXCL13. On the other hand, AhR agonists upregulated Rorc(γt) expression and downregulated CXCR5 expression. These findings suggest that AhR activity and the duration of T cell receptor stimulation contribute to regulating the balance between Tfh and Th17 cell differentiation. Although this in vitro system needs to be further improved, it may be useful for elucidating the mechanisms of Tfh cell differentiation as well as for screening physiological or pharmacological factors that affect Tfh cell differentiation including CXCR5 expression.

Introduction T cell-dependent antibody (Ab) responses have important roles in acquired immunity. Follicular helper T (Tfh) cells play a major role in helping B cells to differentiate into plasma cells in the germinal centers (GCs) of lymphoid organs [1,2]. Tfh cells express the chemokine receptor CXCR5 that contributes to their migration into GCs [3]. The normal formation of GCs requires Tfh cells, and the development and function of Tfh cells are controlled by the transcription factor BCL6 [4][5][6]. Dysregulation of Tfh cell differentiation leads to various disorders including infectious diseases, autoimmune disorders, allergies, and possibly oncogenesis [1].
To clarify the precise molecular mechanisms underlying Tfh cell differentiation, not only in vivo studies but also in vitro studies with proper model systems are needed. Although in vitro models of human Tfh cell differentiation are established [7,8], in vitro mouse models have much room for improvement or replacement [1,9]. Thus, in the present study, we attempted to establish a new in vitro system for inducing the differentiation of mouse naïve CD4 + T cells almost simultaneously into Tfh cells in the absence of other cell types.
Th17 polarization is enhanced by stimulation of the aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor that responds to various polycyclic aromatic hydrocarbons [38][39][40]. AhR agonists can be generated from tryptophan present in the culture medium [41] and enhance Th17 cell differentiation [38][39][40]42]. Furthermore, AhR is functionally upregulated early in the course of T-cell activation [43]. Here we found that addition of an AhR antagonist into the culture further downregulated Rorc(γt) expression and unexpectedly increased CXCR5 expression. The in vitro system that we established in the present study may provide a useful basis for analyzing further detailed mechanisms of Tfh cell differentiation and screening the effects of molecules and conditions on the Tfh cell differentiation.

Cell purification and culture
Naïve CD4 + T cells were purified from spleens by negative selection using EasySep Mouse CD4 + T Cell Enrichment kits (Stemcell Technologies) supplemented with biotinylated mAbs to mouse CD44 (clone IM7) and CD25 (clone PC61) (BioLegend), and subsequently by positive selection with CD62L Microbeads (Miltenyi Biotec). The purity was more than 96%. The purified cells were suspended (1.5-2 × 10 5 cells/ml) in DMEM supplemented with 100 μM non-essential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, 20 mM HEPES (pH 7.2), 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% FCS (cDMEM), and cultured in 96-or 48-well flat-bottom plates (MS-8096R or MS-8048R, Sumitomo Bakelite) coated with anti-CD3 mAb (clone 145-2C11) and other indicated mAbs in the presence of soluble anti-CD28 mAb under 10% CO 2 . Typically, T cells were stimulated with immobilized mAbs to CD3 (3 μg/ml), ICOS (1.5 μg/ml), and LFA-1 (CD11a) (clone M17/4) (1.5 μg/ml) in the presence of soluble anti-CD28 mAb (1 μg/ml) and IL-6 (20 ng/ml) for 3 days. In some experiments, the cells were further cultured in either the same well receiving an equal volume of fresh medium containing the indicated cytokines and Abs or a new culture well coated with or without Abs to CD3, ICOS, and LFA-1 receiving an equal or the indicated volume of medium or replacing the culture supernatant with fresh medium supplemented with the indicated cytokines and blocking Abs. However, when the supernatant of the first culture was carried over to the second culture, further addition of blocking Abs had little effect on Tfh-like cell differentiation. In some experiments, the AhR antagonist CH-223191 or the AhR agonists FICZ or ITE were added to the culture. These reagents were dissolved in DMSO, but final DMSO concentrations were less than 0.1%, and did not significantly affect the results.
In some experiments, RPMI 1640 medium was used in place of the DMEM, and the cells were cultured under 5% CO 2 .

Real-time PCR
Total RNA was isolated from cells using a ReliaPrep RNA Cell Miniprep System (Promega) or RNeasy Mini Kit (Qiagen), and cDNA was generated using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo). cDNA was used as a template for real-time PCR with BrightGreen qPCR MasterMix-ROX (Applied Biosystems) and gene-specific primers. PCR and analysis were performed on a StepOnePlus Real-Time PCR system (Applied Biosystems). The relative expression of each gene was quantified with the 2 −ΔCt value multiplied by 1000, where ΔCt was the difference between the mean Ct value of triplicates or quadruplicates of the sample and that of the endogenous Rplp0 control [44]. Sequences of the primers for Rplp0, Bcl6, Rorc(γt), Prdm1, Tbx21, Gata3, Ahr, and Cyp1a1 are listed in S1 Table in S1 File.

Cytokine ELISA
Cytokine levels in the culture supernatants were analyzed with ELISA kits for IL-21 (R&D Systems).

CFSE labeling and cell proliferation analysis
Naïve CD4 + T cells were labeled with CFSE (5 μM) for 5 minutes at room temperature as previously described [45]. The cells were washed three times and cultured in the Tfh-like cellinducing conditions in the presence or absence of TGF-β1 (0.1 or 1 ng/ml). After the culture, the cells were analyzed for their CFSE fluorescence intensity.

T and B cell co-culture
Indicated helper T cells were incubated with mitomycin C (30 μg/ml; Wako) at 37˚C for 30 minutes. B cells were purified from the spleen of age-and sex-matched C57BL/6 mice by negative selection using EasySep Mouse B Cell Isolation kits (Stemcell Technologies) according to the manufacturer's instructions. Mitomycin C-treated T cells (1.5 × 10 5 cells) were extensively washed and co-cultured with purified B cells (1.5 × 10 5 cells) in the presence of soluble anti-CD3 mAb (0.1 μg/ml) (BioLegend) in 0.2 ml of the medium (RPMI 1640) in 96-well round bottom plates (Falcon) for 7 days. The culture supernatant was analyzed for total IgG Ab production by using IgG (Total) Mouse Uncoated ELISA kits (Invitrogen) according to the manufacturer's instructions. OD450 values (ΔOD450) were calculated by subtracting OD450 readings taken from supernatants from B cells cultured alone from the OD450 values of cocultured samples as previously reported [17]. In some experiments, the cultured cells were stained with anti-CD138 PE, anti-CD45R/B220 APC, and anti-CD4 FITC for excluding CD4 + T cells and detecting plasma cells.

Chemotaxis assay
To examine if the chemokine receptor CXCR5 expressed on T cells was functionally active, transwell chemotaxis assays were performed as previously described [46] with a slight modification. Briefly, 0.6 ml of 10% FCS (RPMI 1640) with or without 1 μg/ml CXCL13 was added into the lower well of the Transwell plates (Corning 3421), and 5 × 10 5 cells suspended in 0.1 ml of 10% FCS (RPMI 1640) were added into the upper well and incubated for 3 hours at 37˚C. The numbers of migrated cells into the lower wells were counted, and their percentages relative to the input cell number were calculated.

Statistical analysis
One-way ANOVA with Tukey-Kramer multiple comparison test or the Student unpaired twotailed t test was used to analyze differences between conditions. Values of p < 0.05 were considered significant. We performed statistical analyses with biological replicates, and "n" represents the number of culture wells or animals.

Blocking of IL-2 signaling enhances the expression of Bcl6 and Rorc(γt) in vitro
To identify the optimal conditions for inducing Tfh cell differentiation in the absence of other cell types in vitro, we stimulated isolated mouse naïve CD4 + T cells with immobilized antibodies to CD3 and various co-stimulatory molecules in the presence of soluble anti-CD28 Ab, IL-6, and neutralizing Abs to some cytokines for 3 days. We found that the combination of immobilized Abs to CD3, ICOS, and LFA-1 and a neutralizing Ab to IL-4 consistently induced the expression of both Bcl6 and Rorc(γt) (Fig 1A and 1B). We also added a neutralizing Ab to IL-2 and blocking Abs to IL-2Rα (CD25) and IL-2Rβ (CD122), as IL-2 is a critical regulator of Tfh cell differentiation [31]. The combination of Abs to IL-2 and IL-2 receptors (IL-2Rs) significantly enhanced the expression of Bcl6 mRNA and BCL6 protein (Fig 1A and 1D), and moderately enhanced Rorc(γt) expression ( Fig 1B). Expression of Prdm1, which encodes the BCL6 antagonist BLIMP-1 was downregulated by anti-IL-2Rs with or without anti-IL-2 ( Fig 1C). For comparison, we activated naïve CD4 + T cells under the Th1, Th2, or Th17 cell-inducing condition for 3 days. These cells expressed significantly lower levels of Bcl6 expression (S2 Fig  in S1 File). These results suggest that strict restriction of IL-2 signaling is indeed crucial for enhancing Bcl6 expression for Tfh-like cell differentiation, but that additional factors or conditions are required to suppress Rorc(γt) expression.

Release from persistent TCR engagement selectively downregulates Rorc (γt) expression
Tfh cell differentiation in vivo appears to be initiated by interactions with antigen-presenting DCs in lymphoid organs and to proceed after detaching from DCs and entering into B cellrich follicles [1]. Accordingly, Powell et al. induced "Tfh0-like" cells after 3 days of stimulation of naïve CD4 + T cells with Abs to CD3 and CD28 in the presence of IL-6 and Abs to IFN-γ and IL-4, followed by 2 days of incubation without the stimulation in the presence of low IL-2 [17]. We hypothesized that release from the first TCR engagement might be required to promote the differentiation. Thus, after 3 days of culture in Ab-coated culture plates (1 st "3d" culture), aliquots of cells were transferred and cultured for 2 more days (2 nd culture) in new plates without immobilized Abs ("3+2d" culture), and other aliquots of cells were continuously cultured in the original wells for 2 more days ("5d" culture) (Fig 2A). Compared with the "3d" culture, the 2-step "3+2d" culture significantly upregulated Bcl6 expression ( Fig 2B) and downregulated Rorc(γt) expression ( Fig 2C). However, the "5d" culture did not downregulate Rorc (γt) expression ( Fig 2C) and upregulated Bcl6 expression (Fig 2B). The Bcl6 expression after the "5d" culture was tended to be lower than that after the "3+2d" culture, although not significantly. The results suggest that the release from the persistent TCR stimulation shifts the balance of differentiation away from the Th17 cell fate toward Tfh-like cell generation.

TGF-β enhances the expression of Bcl6 and Rorc(γt)
TGF-β provides critical additional signals that promote the initial differentiation programs in human Tfh cells, but its effect on mouse Tfh cell differentiation remains controversial [4,7,8,36]. We thus examined the effect of TGF-β1 on the T cell differentiation in the present in vitro system. TGF-β1 significantly enhanced the expression of Bcl6 (Fig 3). The combination of IL-6 and TGF-β1 induces Th17 cell differentiation [48]. Accordingly, TGF-β1 also enhanced Rorc (γt) expression, but the expression was significantly lower after the "3+2d" culture than after the "3d" culture. In contrast, the Bcl6 expression was significantly higher after the "3+2d" culture than after the "3d" culture. TGF-β1 also increased cell number even at 0.1 ng/ml after both the "3d" culture and the "3+2d" culture ( Fig 3C). We thus performed CFSE-based proliferation assays. As shown in S3 Fig in S1 File, we confirmed an enhanced proliferation in TGF- β1-treated cells after the "3d" culture. However, after the "3+2d" culture, CFSE signals were not significantly affected with TGF-β1. The FSC/SSC plots suggested that TGF-β1 suppressed the cell death ( S3A Fig in S1 File). TGF-β1 significantly suppressed ICOS expression after the "3d" culture as reported [7], but only moderately suppressed the expression after the "3+2d" culture ( S3B Fig in S1 File). The results suggest that TGF-β1 potentiates the differentiation, proliferation, and survival of Tfh-like cells.

BCL6 protein expression is not parallel to its mRNA expression
We also analyzed the protein expression of BCL6 and RORγt after the "3d", "3+2d", and "5d" cultures in the presence or absence of added TGF-β1 (1 ng/mL). Surprisingly, the protein expression of BCL6 was not parallel to the mRNA expression (Fig 4A and S4 Fig in S1 File). The protein expression of BCL6 after the "3+2d" culture was apparently lower than that after the "3d" or "5d" culture. On the other hand, the protein expression of RORγt was nearly  Fig 1 in the presence of blocking mAbs to IL-2Rs for 3 days ("3d"). Aliquots of cells were transferred to a new well without Abs to CD3, ICOS, and LFA-1 receiving an equal volume of fresh DMEM supplemented with IL-6 (20 ng/ml), anti-IL-2 (10 μg/ml), and anti-IL-2Rs (10 μg/ml each) and were further cultured for 2 days ("3+2d"). Other aliquots of cells received an equal volume of fresh DMEM supplemented as above in the same well, and further cultured for 2 days ("5d") as schematically shown in (A). After the culture, relative mRNA expression of (B) Bcl6 and (C) Rorc(γt) were analyzed by real-time PCR. Data are presented as mean ± SD of triplicate samples. Results shown are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.
https://doi.org/10.1371/journal.pone.0287746.g002  ("3d"). Aliquots of cells were transferred to a new well without immobilized Abs, receiving an equal volume of fresh DMEM supplemented with IL-6, graded concentrations of TGF-β1, and blocking Abs to IL-2 signaling, and were further cultured for 2 days ("3+2d"). After the "3d" culture (open bar) and the "3+2d" culture (closed bar), relative mRNA expression of (A) Bcl6 and (B) Rorc(γt) was analyzed by real-time PCR. (C) Recovered cell number from the "3d" culture and the "3+2d" culture is shown. Stars indicate that the cell number in the culture with TGF-β1 was significantly different from that in the culture without adding TGF-β1. Data are presented as mean ± SD of quadruplicate samples. Results shown are representative of three independent experiments. **p < 0.01, ***p < 0.001. ns, not significant. https://doi.org/10.1371/journal.pone.0287746.g003

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Tfh-like cell differentiation in vitro parallel to its mRNA expression (Fig 4B and S4 Fig in S1 File). Indeed, the ΔMFI ratio of BCL6 and RORγt expression after the "3+2d" culture was much higher than that after the "3d" or "5d" culture ( Fig 4C). However, the effects of TGF-β1 on both BCL6 and RORγt protein expression were similar to those observed on their mRNA expression (Fig 4 and S4 Fig in S1  File). Interestingly, the FSC intensity after the "3+2d" culture was much lower than that after the "3d" or "5d" culture ( Fig 4C and S4 Fig in S1 File). TGF-β1 further downregulated the FSC intensity. The results suggest that the cells after the "3+2d" culture were much smaller than those immediately after the persistent TCR stimulation. Therefore, after the release from the persistent TCR stimulation, the concentration of BCL6 protein in the cells might not be largely affected, whereas RORγt protein levels might be significantly downregulated. TGF-β1 appeared to enhance the concentration of BCL6 protein as well in these cells. Therefore, we took advantage of the "3+2d" culture condition for improving the in vitro model system in the following experiments. The discrepancy between mRNA expression and protein expression of BCL6 especially after the "3d" culture may be partly due to the repressor function of BCL6 that can repress even its own gene (9).

Tfh-like cells are distinct from Th1 and Th2 cells, but share some features common to Th17 cells
The Tfh-like cells expressed Bcl6 and Rorc(γt), but not the master regulator genes of Th1 and Th2, Tbx21 and Gata3, respectively (S5 Fig in S1 File). Th1 and Th2 cells did not significantly express Bcl6 or Rorc(γt). Th17 cells also exhibited Bcl6 expression but at lower levels than Tfh-like cells and higher levels of Rorc(γt) expression. Tfh cells as well as Th2 and Th17 cells produce IL-21 [27,28,37,49,50]. The present Tfh-like cells produced IL-21, but at lower levels than Th17 cells induced under the present condition ( S5F Fig in S1 File). However, intracellular staining for IL-21 indicated that Tfh-like cells and Th17 cells were producing similar levels of IL-21 after 2 days of CD3/CD28-mediated stimulation (S6 Fig in S1 File). As Tfh-like cells were rested for 2 days before restimulation, there might be a delay in the production of IL-21 compared to Th17 cells. On the other hand, IL-17A production by Tfh-like cells was much less than that by Th17 cells (S6 Fig in  S1 File). These results collectively suggest that the generated Tfh-like cells are distinct from Th1, Th2, and Th17 cells, but still share some features common to Th17 cells.

Replacing the DMEM with RPMI 1640 medium induces the downregulation of RORγt expression
Most of the Tfh-like cells generated at this stage expressed BCL6 protein and their significant population also expressed RORγt protein (Fig 5A). We noticed that replacing the DMEM with RPMI 1640 medium induced the downregulation of RORγt protein and mRNA expression (Fig 5A-5C), but did not significantly affect BCL6 protein and mRNA expression (Fig 5A, 5B, and 5D). Unexpectedly, replacing the medium slightly but significantly upregulated CXCR5 surface protein and mRNA expression (Fig 5E, 5F, and S7A Fig in S1 File). Tfh-like cells generated in the RPMI 1640 medium produced significantly higher levels of IL-21 and lower levels of IL-17A than Th17 cells (S7B and S7C Fig in S1 File), and significantly enhanced IgG production by B cells in vitro (Fig 5I). The IgG production levels were higher than those induced by Th0, Th1, and Th17 cells in T-B cell co-cultures. Flow cytometric analysis of the cultured cells indicated that Tfh-like cells induced CD138 high B220 low plasma cells more efficiently than Th17 cells (S8 Fig in S1 File), while Th0 and Th1 cells induced almost no plasma cells. These results suggest that Tfh-like cells generated in the RPMI 1640 were functional helper T cells.
RPMI 1640 medium contains lower concentrations of aromatic amino acids than DMEM (S2 Table in S1 File). The aromatic amino acids present in culture medium can be or turn to be AhR agonists and enhance Th17 cell differentiation [42]. Thus, replacing DMEM to RPMI 1640 medium might cause reduced AhR activity in the cells. Although expression of Ahr was not significantly affected by changing the medium (Fig 5G), expression of the AhR-regulated gene Cyp1a1 was significantly downregulated (Fig 5H), suggesting that the AhR activity was reduced by the RPMI 1640.

AhR activity regulates the differentiation balance between Tfh-like cells and Th17 cells
Consistent with the above results, adding the AhR antagonist CH-223191 [42,51] to the DMEM culture suppressed Rorc(γt) expression ( Fig 6A) and enhanced Cxcr5 and CXCR5 expression (Fig 6B, 6C, and S9 Fig in S1 File), but did not affect Bcl6 expression (Fig 6D). Expression of PD-1 and ICOS was high and moderately affected by CH-223191 (Fig 6E, 6F,  and S9 Fig in S1 File). In the RPMI culture, CH-223191 further enhanced Cxcr5 and CXCR5 expression, while the AhR agonists FICZ [52] and ITE [53] significantly suppressed the expression (Fig 7A, 7B, and S10A Fig in S1 File). On the other hand, Rorc(γt) expression was

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Tfh-like cell differentiation in vitro significantly suppressed by CH-223191 but upregulated by FICZ (Fig 7C) as expected. ITE tended to upregulate the expression, but not significantly. Bcl6 expression was also not significantly affected in RPMI 1640, and expression of PD-1 and ICOS was moderately or not significantly affected by the AhR modulators (Fig 7D-7F, and S10 Fig in S1 File).

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CXCR5 expression slightly but significantly increased after the 2-step "3+2d" culture compared with that after the first "3d" culture or the "5d" culture in the RPMI medium (Fig 8A  and S11 Fig in S1 File). Significant CH-223191-dependent upregulation of CXCR5 expression was observed after the "3+2d" culture, but not after the first "3d" culture or the "5d" culture ( Fig 8A). The upregulation was observed only when CH-223191 was added in the first culture ( Fig 8B). These results suggest that AhR activity prohibits the increase in CXCR5 expression after the release from TCR engagement, but that AhR-dependent signals during TCR engagement are responsible for the prohibition.
To test if the CXCR5 molecules expressed on Tfh-like cells generated in the presence or absence of CH-223191 in RPMI 1640 were functionally active, we performed transwell  Fig 2 ("3d", "3+2d", and "5d") but in RPMI medium containing TGF-β1 (1 ng/ml). CH-223191 (5 μM) or vehicle control was added in the first culture. The supernatant of the first culture was transferred to the second culture in a new culture plate with ("5d" culture) or without ("3+2d" culture) immobilized mAbs to CD3, ICOS, and LFA-1. An equivalent volume of fresh migration assays. The CH-223191-treated cells exhibited significant chemotaxis toward the CXCR5 ligand CXCL13 (Fig 8C), whereas FICZ-treated cells failed to enhance chemotaxis toward CXCL13 (S12A Fig in S1 File). Control cells tended to migrate toward CXCL13, although statistically not significant (Fig 8C and S12 Fig in S1 File). These results suggest that CXCR5 expressed on the CH-223191-treated Tfh-like cells was functionally active, and that regulation of the AhR activity affects their chemotactic capacity. We also confirmed that the CH-223191-treated cells could produce IL-21. Although CH-223191 at 5 μM moderately suppressed IL-21 production, CH-223191 at 2.5 μM suppressed neither IL-21 production nor Bcl6 expression but significantly suppressed both Rorc(γt) expression and IL-17A production. It also enhanced chemotaxis toward CXCL13 (S12D Fig in S1 File).
CCR7 and CXCR4 are expressed on naïve T cells and downregulated during differentiation to effector T cells [54]. The expression levels of CCR7 and CXCR4 in the in vitro-generated cells were low and not affected by CH-223191 (S13 Fig in S1 File). Expression of CXCR4, but not CCR7, was downregulated after the "3+2d" culture compared with the "3d" culture. In sum, these results suggest that AhR activity regulates the differentiation balance between Tfhlike cells and Th17 cells.

The CH-223191-treated Tfh-like cells do not express RORγt but exhibit BCL6 protein levels equivalent to Tfh cells in vivo
We examined protein expression of BCL6 and RORγt in CH-223191-treated Tfh-like cells. The most cells expressed BCL6 but did not express RORγt (Fig 9A). The expression levels of BCL6 and PD-1 were nearly equivalent to or even higher than those in Tfh cells from mesenteric lymph nodes and spleens of immunized mice (Fig 9B, Fig 9C, and S10 and S14 Figs in S1 File). However, the average expression levels of CXCR5 in CH-223191-treated Tfh-like cells appeared to be still lower than those in Tfh cells from immunized mice (S14E Fig in S1 File). These results suggest that Tfh-like cells generated at this stage well resemble Tfh cells in vivo but that additional factors may be required for higher expression of CXCR5.

Discussion
To clarify the kinetic and molecular mechanisms of Tfh cell differentiation, we established an in vitro model system of mouse Tfh cell differentiation in the absence of other cell types. Upon TCR/CD3 stimulation of naïve CD4 + T cells in vitro, costimulation with immobilized mAbs to ICOS and LFA-1 and a soluble mAb to CD28 consistently led to enhanced expression of Bcl6, the master regulator gene for Tfh cell differentiation. ICOS signaling is essential for upregulating BCL6 expression through nuclear exclusion of FOXO1, and BCL6 upregulates Tfh marker molecules such as CXCR5, PD-1, and ICOS, and downregulates CCR7 [21,24,55]. LFA-1 stimulation also enhances BCL6 expression in a Talin-1-dependent manner [22]. BCL6 represses expression of microRNAs and Id2, which can suppress the expression of Tfh-specific medium containing IL-6 (20 ng/ml), TGF-β1 (1 ng/ml), anti-IL-2 (10 μg/ml), and anti-IL-4 (10 μg/ml) was added. (B) Naïve CD4 + T cells were cultured as in (A) ("3+2d" culture) except that culture supernatants of the first "3d" culture were removed and resuspended in twice the volume of fresh RPMI medium containing TGF-β1 (1 ng/ml), IL-6 (20 ng/ ml), and mAbs to IL-2, CD25, and CD122 (10 μg/ml each), and the cells were further cultured in new culture plates for 2 days. CH-223191 (5 μM) was added in the first or second culture or both. (A and B) Data are presented as mean ± SD of triplicate samples. Results shown are representative of three independent experiments. (C) Naïve CD4 + T cells were cultured as in (A) ("3+2d" culture). CH-223191 (5 μM) or vehicle control was added in the first culture. The cells were assessed for chemotactic activity toward the chemokine CXCL13 (1 μg/ml) in transwells. Data are presented as mean ± SD of quadruplicate samples. Results shown are representative of five independent experiments. **p < 0.01, ***p < 0.001. ns, not significant. https://doi.org/10.1371/journal.pone.0287746.g008

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genes and inhibit Tfh cell differentiation [6,56,57]. BCL6 also represses the development of other Th lineages by directly suppressing the promoter activities of the Tbx21 and Rorc genes or by posttranscriptional regulation of Gata3 in Tfh cells [4,58]. BCL6 represses its own gene as well, while it indirectly induces the expression of several other genes by repressing repressors [9,57]. The BCL6 and E3 ligase Cullin3 complex represses Bcl6 and Batf, which are involved in activating the Bcl6 promoter, and thus exerts negative feedback on the Tfh program [59]. Upregulation of Bcl6 expression after the release from persistent TCR engagement in vitro might involve release from this negative feedback mechanism. Interestingly, the flow cytometric analysis of intracellular BCL6 protein revealed that the apparent BCL6 protein expression after the release from TCR engagement was downregulated unlike the Bcl6 mRNA expression. However, the cell sizes after the release, especially in the presence of added TGF-β1, appeared to be much smaller than those right after the TCR engagement, suggesting that BCL6 protein concentration might not be significantly downregulated after the release. Indeed, significant levels of BCL6 protein, as well as its mRNA expression, were detected in after the release from TCR engagement in the presence of TGF-β1. Alternatively, STAT activities might contribute to this phenomenon. TCR stimulation can induce delayed and prolonged activation of STAT5 as well as STAT3 [60]. In addition, persistent TCR activation may enhance IL-6-dependent production of IL-21, which can activate not only STAT3 but also STAT5. IL-6 and IL-21 induce BCL6 upregulation depending on STAT3 activation [10,12], whereas STAT5 outcompetes STAT3 for binding to a regulatory region of the Bcl6 gene and inhibits Bcl6 expression [61].
TGF-β signals promote Tfh cell differentiation in human CD4 + T cells but their role in mouse Tfh cell differentiation has remained controversial [4,7,8,36]. Our results suggest that TGF-β1 enhances the differentiation, proliferation, and survival of mouse Tfh-like cells in our culture system. The strict regulation of IL-2 signals with blocking Abs to both IL-2 and IL-2Rs might contribute to the TGF-β1-induced enhancement. We mainly used 1 ng/ml TGF-β1 in the present study. Higher concentrations (5 or 10 ng/ml) of TGF-β1 induced higher levels of Bcl6 expression than 1 ng/ml TGF-β1. TGF-β suppresses IL-2Rα expression and STAT5 activation [36]. Accordingly, our preliminary experiments revealed that lower concentrations of anti-IL-2 and anti-IL-2R Abs were sufficient to enhance Bcl6 expression in the presence of higher concentrations of TGF-β1. When blocking of IL-2 signaling was insufficient, the combination of TGF-β and residual IL-2 signals might induce regulatory T cells that suppress Tfh cell differentiation. However, we could not detect Foxp3 expression under the present condition. The sensitivity of CD4 + T cells to IL-2 or TGF-β may differ between the two species under their culture conditions.
Cxcr5 expression was not synchronously regulated with Bcl6 expression. BCL6 is required for maintaining CXCR5 expression in T cells, but not for the expression in the early phase of the immune response [62]. We found that CXCR5 expression was enhanced after the release from persistent TCR engagement in the presence or absence of the AhR antagonist CH-223191. Expression of some other chemokine receptors including CXCR3 and CCR9, is also enhanced after the release from persistent TCR engagement in the presence of IFN-γ and retinoic acid, respectively [63,64]. Higher doses of antigen and higher strength of TCR are considered to favor the induction of Tfh cell differentiation [65]. It may suggest that longer duration favors Tfh cell differentiation. However, medium-affinity TCRs bias mouse naïve T cells to become Tfh cells, while higher-affinity TCRs promote the formation of Th1 or Th17 cells [66]. Therefore, although persistent TCR engagement is likely to be required for Tfh cell differentiation, the optimal duration of TCR engagement for Tfh cell differentiation may vary depending on the nature and dose of the antigen and other factors.
AhR activity appeared to be critical for the differentiation balance between Tfh-like cells and Th17 cells. It is well known that AhR activity contributes to the Th17 cell differentiation [38][39][40], while its effects on Tfh cell differentiation varied depending on the agonist used in vivo [67]. A recent study in vivo indicated that AhR indeed modulates Tfh cell responses to influenza A virus infection in mice [68]. We found in our in vitro system that Rorc(γt) expression was upregulated by AhR agonists and downregulated by the antagonist, and that Cxcr5 expression but not Bcl6 expression was reciprocally regulated by these AhR modulators. Even by changing the medium from DMEM to RPMI 1640, which contains lower concentrations of aromatic amino acids, we observed the reciprocal regulation of Rorc(γt) and Cxcr5 expression. The AhR activity during TCR engagement appeared to be responsible for suppressing CXCR5 expression after the release from TCR engagement. It remains unclear how AhR activity could be repressed in vivo. However, competition between AhR and other transcription factors such as hypoxia-inducible factor HIF-1α for binding to aryl hydrocarbon receptor nuclear translocator (ARNT) might contribute to the repression of AhR activity, since their transcriptional activities depend on the formation of heterodimers with ARNT [69]. HIF-1α depletion from CD4 + T cells reduces frequencies of antigen-specific GC B cells, Tfh cells [70].
Efficient induction of surface CXCR5 expression in mouse T cells in vitro has been difficult at least in the absence of antigen presenting cells [13,14]. However, we could induce CXCR5-bearing Tfh-like cells that were capable of chemotaxis toward CXCL13. Similar levels of CXCR5 expression can be observed in a significant population of Tfh cells in vivo, but the mean CXCR5 expression levels in Tfh cells in vivo were significantly higher. Other factors may also contribute to the upregulation of CXCR5 expression in vivo. Furthermore, as Tfh cell differentiation appears to be a multi-step process and requires cognate interaction with B cells after the initial interaction with DCs [1,9], the secondary TCR-mediated stimulation may be required to induce full maturation of the Tfh-like cells and higher CXCR5 expression in them in vitro.
The Tfh-like cells generated especially in DMEM without AhR antagonists might at least partially resemble Tfh17 cells, a subset of Tfh cells expressing both RORγt and BCL6 [71], although their expression of CXCR5 was low. Circulating Tfh (cTfh) cells, first found in human tonsils and blood, share the phenotype and functional nature of bona fide Tfh cells in GCs [71][72][73]. While Tfh cells as well as cTfh cells appear to be heterogenous in their phenotypes and functions reminiscent of conventional helper T cells, including Th1, Th2, and Th17 cells, the mechanism underlying their differentiation remains unclear [72,73]. Multiple pathways are likely involved in the induction of Tfh cell subsets. In the differentiation of Tfh1-like cells, however, IL-12 signaling can be involved [15,17]. Tfh17-like cells represent the most predominant subset in severe COVID-19 cases [74]. Regulation of the development of Tfh and cTfh cell subsets might change the disease outcome.

Conclusion
We generated murine Tfh-like cells in vitro in the absence of other types of cells by stimulating naïve CD4 + T cells via CD3, ICOS, LFA-1, and CD28 in the presence of IL-6 and TGF-β, and blocking IL-2 signaling, followed by release from persistent TCR/CD3-mediated stimulation. These cells exhibited the fundamental key features of Tfh cells, including the expression of BCL6, PD-1, ICOS, and functional CXCR5 even if not high enough, and produced IL-21 and exhibited helper T cell activity. Suppression of AhR activity enhanced Tfh cell differentiation. The present culture system may provide a useful in vitro system for further analysis of the differentiation mechanisms of Tfh cells and Tfh subsets and for screening for modulators of their differentiation including CXCR5 expression.